CH637628A5 - Peptide having therapeutic activity - Google Patents

Description

The present invention relates to new, therapeutically valuable peptides and acid halides, optionally N-substituted acid amides, esters and N-acylates, pharmaceutically acceptable acid addition salts and basic salts of these peptides.

The therapeutically useful peptides of the present invention contain at least four amino acid residues and can contain twelve, thirteen or more such residues.

The products according to the invention are useful therapeutic agents because of their ability to contract smooth muscles, release histamine from mast cells and to increase the permeability of the vessels. Certain compounds according to the invention are useful, as explained in more detail below, because of their opposite effect. In particular, certain peptides of the present invention are agonists or promoters, while others are antagonists or inhibitors. Some are useful for the treatment of cardiac arrhythmia.

The carboxyl end of the peptide is formed by an amino-substituted a-amino acid, such as arginine or canavanine. For simplification, this end link is referred to below as arginine. The arginine is linked with a peptide bond via its a-amino group to the carboxyl group of a lower aliphatic amino acid, which expediently contains up to four amino acid members. The currently preferred second amino acid is glycine or alanine.

The third amino acid in the peptide is linked via its carboxyl group to the amino group of the second amino acid

40 den. This third amino acid is a hydrophobic amino acid, e.g. Isoleucine, leucine, valine, methionine, phenylalanine, tyrosine or tryptophan. Leucine is currently preferred because it is readily available in both D and L forms and contributes to the effectiveness of the peptide in a satisfactory manner.

The identity of the fourth amino acid does not appear to be critical, but a fourth amino acid must be present to achieve the useful activity. Virtually any of the 20 common amino acids appears to be useful for this grouping. Compounds with the highest activity are those in which the fourth amino acid is glycine or glutamine. However, other amino acids, such as alanine, asparagine, serine, tyrosine or phenylalanine, can easily be used as the fourth amino acid.

The presence of a fifth, preferably hydrophobic, amino acid, e.g. B. leucine, noticeably increases the activity of the peptide. Other hydrophobic amino acids, e.g. those mentioned above are also useful.

6o It was observed that the activity increases with a length of the peptide chain up to a length of thirteen amino acids. However, the cost of producing polypeptides with considerably more amino acid members does not appear to be justified by the modest improvements that can be achieved.

Those compounds of the present invention in which the first amino acid is arginine are agonists.

If the arginine via its carboxyl group to the amino

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group of a lower aliphatic amino acid, such as. B. glycine, proline, alanine or serine, the resulting products are antagonists. At low doses, they prevent smooth muscle contraction, the release of histamine and the permeability of the vessels. The amino acid to which the arginine is attached could be referred to as an inhibitor amino acid. The currently preferred inhibitor amino acid is glycine because it is readily available and can be incorporated into the molecule without the introduction of a new asymmetry center.

It is also possible to modify the activity of the peptides of the present invention in other ways, e.g. using a D-amino acid as part of the peptide instead of the naturally occurring L-amino acids. Various products in which L-arginine is replaced by D-arginine resist the hydrolytic activity of carboxypeptidase-B. The product is therefore stable over an extended period of time and can be used to produce long-term dosage forms. The same result can also be achieved by substituting proline in the second position.

Owing to their amphoteric nature, the compounds according to the invention can be used in the form of pharmaceutically acceptable salts, which can be either metal salts or acid addition salts. These salts have the advantage of water solubility and are particularly useful for parenteral administration. The metal salts, especially the alkali metal salts, are relatively stable and for this reason are often preferred to the acid addition salts. The sodium salts are particularly preferred because they are easy to manufacture.

The acids which can be used to prepare the acid addition salts of the peptides according to the invention are those acids which contain non-toxic anions, such as, for. B. hydrochloric, sulfuric, phosphoric, vinegar, lactic, lemon, tartaric, oxalic, succinic, maleic, gluconic, saccharic and similar acids. A large number of non-toxic derivatives of the polypeptides according to the invention can thus be used. For example, the products, particularly those inhibitors with a terminal glycine carboxyl group, can be reacted with thionyl chloride to form the acid chloride, and the latter can be reacted with ammonia to form an amide. Amines naturally form N-substituted amides.

Other useful derivatives can be obtained by modifying the free functional groups on the peptide backbone. For example, free hydroxyl groups and free amino groups can be easily converted. A convenient class of derivatives is that in which a free hydroxyl group, which may be aliphatic or aromatic in nature, is esterified with an alkanoyl or alkenoyl group, which may contain up to 18 carbon atoms or more. On the other hand, a free amino group with alkanoyl or alkenoyl groups which contain up to about 18 carbon atoms can be acylated. In both cases, the preferred derivatives are those in which the alkanoyl or alkenoyl groups contain 11 to 18 carbon atoms, because longer hydrocarbon chains impart increased lipid solubility to the molecules and thereby increase their transport through cell barriers.

The compounds according to the invention can be prepared in various ways as are known for the synthesis of simple and complex polypeptides and even of proteins with a relatively low molecular weight. In general, these methods involve step-by-step synthesis by successive addition of the amino acids to gradually produce larger molecules. The amino acids are bound to one another by condensation between the carboxyl group of one amino acid and the amino group of another amino acid to form a peptide bond. To control these reactions, it is useful to block the amino group of one acid and the carboxyl group of the other acid. The blocking groups should be chosen so that they can be easily removed without affecting the polypeptide by racemization or by hydrolysis of the peptide bonds formed. Certain amino acids have additional functional groups, e.g. B. the hydroxyl group of tyrosine or threonine. In general, it is necessary to also block these additional groups with an easily removable blocking agent so that they do not interfere with the condensation desired to form peptide bonds.

A large number of methods for the synthesis of polypeptides and a large number of blocking agents are known to those skilled in the art. Most of these methods can be applied to the polypeptides which form the subject of the present invention. There is no point in describing the use of all of these methods here. Preferred methods are mentioned in the following examples.

The currently preferred method for the synthesis of the products according to the invention is the Merrifield method. In this method, an amino acid is bound to a resin particle in the form of an ester bond, and the peptide is generated in stages by the successive addition of protected amino acids to the growing chain. The process is well known and has been described in numerous articles; see e.g. B.W. Erikson and R.B. Merrifield (1976) Solid phase peptide synthesis in "The Proteins", Neurath and Hill, Academic Press, New York, pages 255 to 527.

For simplification, the usual abbreviations for the different amino acids are used in the following description. They are all known to those skilled in the art, but some of the important amino acids are listed below for safety:

Arginine - Arg

Alanine - Ala

Leucine - Leu

Glycine - Gly

Histidine - His

Serine - Ser

Glutamine - gin

As indicated above, the agonists of the present invention increase smooth muscle contraction, increase histamine release, and increase vascular permeability. They are therapeutically useful for inducing inflammation response in humans and mammals that are deficient in this regard. So they can be used to help the body create its defense mechanism against the ingress of harmful microorganisms or other overstressing (stress). The antagonists are clinically useful for local or systemic episodic or traumatic smooth muscle contraction, histamine release, or increased vascular permeability. For example, they can be used in nasal aerosols during asthma attacks or bronchial allergy. They can be used as prophylactics for such syndromes as shock in dengue fever.

The compounds according to the invention are useful therapeutic agents for humans and mammals and are already effective for stimulation in extremely low amounts5

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inhibition or inhibition. The doctor or veterinarian will determine the dosage, which is the cheapest for a particular application. This dosage can vary from one patient to another depending on the size of the patient, the condition to be treated and other factors which are easy to assess by a person skilled in the art. The products can be administered in very high amounts, even up to 2 or more grams per day. They are usually supplied in dosage units which contain up to about 250 mg of the active ingredient. The number of units to be administered daily can be recorded by prescription and varies depending on the condition to be treated.

The compounds can be used alone

however, they are generally administered together with pharmaceutically acceptable, non-toxic carriers, the amount of which is determined by the suitability and chemical nature of the carrier, the mode of administration chosen and standard pharmaceutical practice. For intravenous and intramuscular administration, they can be used in the form of a sterile solution containing other solutes, e.g. B. enough table salt or glucose to make the solution isotonic. For topical administration they can be combined with suitable carriers, e.g. Petrolatum or other inert semi-viscous materials. Dosage units for oral administration can be used in the form of tablets or capsules containing excipients such as e.g. Starch, milk sugar, certain tones, etc. included.

Although arginine is currently the preferred first amino acid, other a-amino acids, e.g. Ca-navanin, also useful. Due to the presence of the Q-amidino group [-C (= NH) -NH2] all can be considered analogs of arginine. They can have a larger or smaller number of methylene or imino groups in the side chain bonded to the a-carbon atom. One or more of these groups can be replaced by another group, such as an oxi, thio, imino or carbonyl group. One or more hydrogen atoms bonded to carbon or nitrogen can be replaced by an alkyl group, which typically contains up to 6 carbon atoms, but preferably methyl or ethyl. As mentioned above, the acid can be in the D or L form.

The following examples serve to illustrate the present invention.

example 1

Synthesis of L-alanyl-L-seryl-L-histidyl-L-leucyl-glycyl-L-leucyl-L-alanyl-L-arginine

Preparation of Na-tert-butyloxycarbonyl-Ns- (4-toluenesulfonyl) -L-arginyl-oxymethyl- (styrene-copoly-1% -divinyl-benzene) - (II). A solution of Na-tert-butyloxycarbonyl-Ng- (4-toluenesulfonyl) -L-arginine (1.3 g) in ethanol (15 ml) and water (1.35 ml) was added by adding a solution of cesium bicarbonate (0 , 43 g) in water (1.35 ml) brought to pH 7.1. The solution obtained was evaporated to dryness at 40 ° C. under reduced pressure. Benzene (25 ml) was added and the mixture evaporated to dryness under the same conditions; this procedure was repeated four more times. The resulting solid was dried at a pressure of 1 torr for 2.5 hours to give the cesium salt of Boc-Arg (Tos) (I) as a white solid (1.7 g). Chloromethyl (styrene-copoly-1% -di-vinylbenzene) (3.5 g; 0.23 millimole Cl per gram of polystyrene) was swollen in N, N-dimethylformamide (DMF; 15 ml) and mixed with a solution of the salt I (1.7 g) mixed in DMF (10 ml). The mixture was mechanically stirred for 75 hours and kept at 50 ° C. A solution of salt I (3.7 g) in DMF (25 ml) was added and the mixture was stirred for 95 hours and kept at 50 ° C. The resin was collected on a sintered glass filter and washed successively with DMF (three 25 ml portions), absolute alcohol (three 25 ml portions) and dichloromethane (three 25 ml portions). After drying at 1 torr for 2 hours, Resin II (3.3 g) contained 0.17 millimoles of arginine per gram of polystyrene, as by treating 10 mg portions with 10% anisole in liquid HF for 30 minutes at 0 ° C, hydrolysis of the acid-soluble material with 6N HCl for 24 hours at 110 ° C and amino acid analysis of the hydrolyzate was determined.

Preparation of Na-tert-butyloxycarbonyl-L-alanyl-O -benzyl-L-seryl-Nim- (4-toluenesulfonyl) -L-histidyl-L-leucyl-glycyl-L-leucyl-L-alanyl-NE- ( 4-toluenesulfonyl) -L-argmyl-oxymethyl- (styrene-copoly-1% -divinylbenzene) - (III). The Boc-Arg (Tos) -Har ^ -II (2.0 g; 0.34 millimole arginine) was swollen in dichloromethane (40 ml) in a 75 ml glass reaction vessel equipped with a sintered glass plate. The resin was filtered by applying air or nitrogen pressure to the vessel and / or reduced pressure to the outlet opening located below the sintered disk until most of the solution was removed.

A. The protecting groups were removed by adding a 1: 1 (v / v) solution of trifluoroacetic acid-dichloromethane (40 ml) to the reaction vessel, stirring the mixture for 1 minute and filtering as before. Then a second portion of the 1: 1 (v / v) mixture of tri-fluoroacetic acid and dichloromethane (40 ml) was added and the mixture was mixed for 30 minutes and filtered.

B. Washing was done in a similar manner by washing the resin sequentially with dichloromethane (for 40 ml portions for 1 minute each), with 2-propanol (two 40 ml portions for 1 minute each) and with dichloromethane (five 40 -ml servings for 1 minute each).

C. Neutralization was carried out by washing the resin with 1:19 (v / v) ethyldiisopropylamine-dichloromethane (three 40 ml portions for 2 minutes each).

D. Washout step B was repeated.

E. The coupling was carried out by adding a dichloromethane solution (10 ml) of the desired Boc-amino acid (4 millimoles per millimole of the original arginine) to the resin, mixing for 2 minutes, adding a dichloromethane solution (10 ml) of N, N '-Dicyclohexylcarbodiimide (3 millimoles per millimole of original arginine) to mix, mix for 30 minutes and filter.

F. Washout step B was repeated.

G. Neutralization level C was repeated.

H. Washout step B was repeated.

I. Coupling stage E was repeated.

J. Washout step B was repeated.

Steps A through J performed a cycle of synthesis during which a protected amino acid residue was added to the peptide. This cycle was initially carried out with Boc-L-alanine and then successively with Boc-L-leucine, Boc-glycine, Boc-L-leucine, Boc-nini- (4-toluenesulfonyl) -L-histidine, Boc-OP-Ben -Zyl-L-serine and Boc-L-alanine, in which "Boc" means "Na-tert-butyloxycarbonyl".

Deprotection and purification of L-alanyl-L-seryl-L-histidyl-L-leucyl-glycyl-L-leucyl-L-alanyl-L-arginine (IV). A portion of the protected octapeptide resin III (0.26 g) was treated with 10% anisole in liquid HF (6 ml) for 1 hour at 0 ° C. After evaporating the HF with a water jet pump and then with a vacuum pump, the resin was washed with ether (three 2 ml portions) to remove the rest

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Remove anisole and use trifluoroacetic acid (five 2 ml portions) to remove the peptide IV. After evaporation of the trifluoroacetic acid, the residue was dissolved in 5% aqueous acetic acid and lyophilized to give the peptide, which was dissolved in 1% aqueous acetic acid (2 ml) and subjected to gel filtration on a 2.5 x 100 cm column from «Bio -Gel P-2 »(100 to 200 mesh, Bio-Rad) subjected, which was pumped through with 1 percent acetic acid in an amount of 23 ml per hour. The main ninhy-positive fraction (175 to 300 ml) was collected and lyophilized. A portion of the residue (19 mg) was dissolved in 0.01 M NaCl, 0.01 M NaH2P04 (pH 4.5; 1.5 ml) and placed on a 1 x 50 cm column made of carboxymethyl cellulose («Cellex CM », Bio-Rad), through which a linear salt gradient of 0.01 to 0.20 M NaCl (both solutions 100 ml, 0.01 M NaH2P04, pH 4.5) is pumped in an amount of 30 ml per hour has been. The major peptide fractions (130-170 ml), determined by ultraviolet absorption at 206 nm, were collected and lyophilized. The remaining mixture of salt and peptide was dissolved in 1% acetic acid (2 ml) and on a 1.5 x 85 cm column of "Bio-Gel P-2", through which 1% acetic acid in an amount of 15 ml per hour was pumped through, desalt. The major peptide fraction (50-75 ml), determined by absorbance at s 206 nm, was collected and lyophilized to obtain the pure octypeptide IV (10 mg). This material gave the expected amino acid analysis (see Table I) and amino acid sequence as determined by automated Edman degradation. It also has a single nin-io hydrine-positive and Pauly-positive flock in thin-layer chromatography (Rf 0.73 against valine at Rf 0.64) in 15: 12: 10: 3 (volume) 1-butanol-water-pyridine vinegar acid and in high voltage electrophoresis (RArg 0.76 at pH 5.0 and RArg 0.58 at pH 6.4, where RArg is the distance i5 between the centers of the peptide IV and valine spots divided by the distance between the Centers of arginine and valine stains means).

Each of the remaining 20 peptides mentioned in Tables I or II was prepared in essentially the same way.

Table I

No amino acid sequence

Amino acid composition (molar ratios) His Arg Ser Glu Gly Ala

Leu

TIC (Rr)

Efficacyb (nmol)

1

Gly-Leu-Ala-Arg

0

1.04

0

0

1.08

10.9

1.00

0.73

1200-1400

2nd

Leu-Gly-Leu-Ala-Arg

0

0.87

0

0

1.12

1.12

2.0

0.80

150-300

3rd

His-Leu-Gly-Leu-Ala-Arg

0.98

0.97

0

0

1.18

1.09

1.00

0.79

10-20

4th

Ser-His-Leu-Gly-Leu-

Ala-Arg

0.99

1.00

0.93

0

1.06

1.05

2.00

0.77

6-10

5

Ala-Ser-His-Leu-Gly

Leu-Ala-Arg

0.95

1.00

0.97

0

1.13

1.94

1.94

0.73

5-8

6

CH3CO-Ala-Ser-His-Leu-

Gly-Leu-Ala-Arg

0.90

1.00

0.98

0

1.32

2.00

2.33

0.81

8-12

7

Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-

Leu-Ala-Arg

1.97

3.00

1.04

1.10

1.11

3.30

2.18

0.71

4-7

8th.

Ala-Ser-His-Leu-Gly

Leu-Ala-Arg-Gly

0.90

1.00

0.97

0

2.20

1.92

2.09

0.73

280-560

9

Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-

Leu-Ala-Arg-Gly

1.90

3.00

0.94

0.96

2.30

3.09

2.20

0.71

240-360

"Ratio of peptide mobility to solvent front migration on silica gel thin-layer plates in 15: 12: 10: 3 (volume) 1-butanol-water-pyridine-acetic acid.

b Amount of peptide per 100 ml of solution required to completely contract strips of guinea pig ileum with Tyrode solution.

Table II

No. Amino Acid Sequence Amino Acid Composition Thin Layer Dataa Ileum

(Molar ratios) (Rfxl00) effectiveness b

Gly Leu Ala Arg A B C D (nmol)

10 Ala-Ala-Ala-Leu-Gly-Leu-

Ala-Arg

1.03

2.00

4.04

1.06

16

45

56

-

6-8

11

Ala-Ala-Leu-Gly-Leu-Ala-Arg

1.02

2.00

3.06

1.08

16

45

60

-

10-14

12

Ala-Leu-Gly-Leu-Ala-Arg

1.03

2.00

1.99

1.05

22

50

65

-

5-11

13

Asn-Lys-Pro-Leu-Gly-Leu-

Ala-Arg

1.03

2.00

1.01

1.03d

-

17th

30th

-

14-27

14

CHjCO-Leu-Gly-Leu-Ala-Arg

1.03

2.00

1.02

1.03

-

66

66

-

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Table II (continued)

No Ammo Acid Sequences / Amino Acid Composition Thin Layer Data "ileum

(Molar ratios) (Rfxl00) effectiveness "

Gly Leu Ala Arg A B C D (nmol)

15 CH3CO-His-Leu-Gly-Leu-

Ala-Arg

1.05

2.00

1.01

1.03e

63 4:

8 13

8-17

CH3CO-Ala-Leu-Gly-Leu-

Ala-Arg

0.98

2.00

1.98

0.91

42

18th

19-38

CH3CO-Ala-Leu-Gly-Leu-

Ala-Cane

0.99

2.00

1.98

45

21st

86-170

CH3CO-Ala-Ala-Ala-Leu-

Gly-Leu-Ala-Arg

1.21

2.00

4.27

1.02

39

10th

7-13

a Ratio of peptide mobility to solvent front migration on silica gel thin-layer plates in the following solvent systems (volume ratio): A: 4: 1: 1 1-butanol-acetic acid-water; B: 1: 1: 1: 1 1-butanol-ethyl acetate-acetic acid-water; C: 5: 5: 1: 3 ethyl acetate pyridine acetic acid water; D: 16: 2: 1: 1 1-butanol-pyridine-acetic acid-water.

b Required amount of peptide / 100 ml Tyrode solution for complete contraction of a strip of guinea pig ileum.

Q Also: His, 0.96.

d Also: Asn, 1.00; Lys, 1.02; Per, 1.01.

c L-canavanine (Can) has a side chain [CH2-CH2-0-NH-C (= NH) -NH2], similar to that of arginine (Arg), except that an oxygen atom replaces the delta methylene group.

S

Claims (18)

637 628 2nd PATENT CLAIMS 1. peptide with therapeutic activity, which contains at least four amino acids, characterized by the presence of an amido-substituted a-amino acid as the first amino acid at the carboxyl end, this first amino acid being bound with a peptide compound via its amino group to the carboxyl group of a second amino acid, which consists of glycine or alanine, and which second amino acid is linked with a peptide bond via its amino group to the carboxyl group of a third hydrophobic amino acid, which third amino acid in turn is linked with a peptide bond via its amino group to the carboxyl group of a fourth amino acid, and the acid halides, optionally N- substituted acid reamides, esters and N-acylates, pharmaceutically acceptable acid addition salts and basic salts of these peptides. 2. Peptide according to claim 1, characterized in that the fourth amino acid consists of glycine, glutamine, alanine, asparagine, serine, tyrosine or phenylalanine. 3. Peptide according to claim 1 or 2, characterized in that the amino-substituted a-amino acid is arginine and the peptide optionally contains a fifth, preferably hydrophobic, amino acid. 4. Peptide according to claim 3, characterized in that the second amino acid is alanine and the fourth amino acid is glycine. 5. Peptide according to claim 3, characterized in that the second and fourth amino acids are both glycine. 6. Peptide according to claim 3, characterized in that the second amino acid is glycine and the fourth amino acid is glutamine. 7. Peptide according to claim 3, characterized in that the second amino acid is alanine and the fourth amino acid is glutamine. 8. Peptide according to claim 3, characterized in 5 net that the arginine is D-arginine. 9. Gly-Leu-Ala-Arg according to claim 1. 10. Leu-Gly-Leu-Ala-Arg according to claim 1. 11. His-Leu-Gly-Leu-Ala-Arg according to claim 1. Ser-His-Leu-Gly-Leu-Ala-Arg according to claim 1. 10 13. Ala-Ser-His-Leu-Gly-Leu-Ala-Arg according to claim 1. 14. CH3CO-Ala-Ser-His-Leu-Gly-Leu-Ala-Arg according to claim 1. 15. Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu- 15 Ala-Arg according to claim 1. 16. A peptide according to claim 1, characterized in that the amino-substituted a-amino acid with a peptide bond via its carboxyl group to the amino 20 group of an amino acid is bound, which consists of glycine, proline, alanine or serine. 17. Peptide according to claim 2, characterized in that the amido-substituted a-amino acid with a peptide bond via its carboxyl group to the amino 25 group of an amino acid is bound, which consists of glycine, proline, alanine or serine. 18. Peptide according to claim 17, characterized in that the amino acid is glycine. 30 19. Ala-Ser-His-Leu-Gly-Leu-Ala- Arg-Gly according to claim 16. 20. Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu-Ala-Arg-Gly according to claim 16.